RU1837773C - Strain of bacteria bacillus thuringiensis var kurstaki for production of insecticidal preparation - Google Patents

Abstract

The invention provides a culture of a sporogenic, crystalliferous mutant of Bacillus thuringiensis having the identifying characteristics of a strain selected from NRRL B-18195, NRRL B-18196 and NRRL B-18197, wherein the strain is characterised as having the ability to preoduce bypyramidal crystals composed of toxic protein and requiring a leucine and valine containing nutrient medium for growth, sporulation and crystal production. The cultures are useful as biocontural agents agents for controlling Leipdopteran insects.

Description

The invention relates to the preparation of a new insecticidal preparation based on a new strain of Bacillus thuringlensis Varo mdrrisonl NRRL B-18196.

The insecticidal preparation is used to control the insect species of the order Squamous echinidae, in particular, TRICHOPLUSIANI, cabbage scoop and ARTOGEIA RAPAE and porto mermitids.

The spore-forming microorganism B.:huringiensls was isolated over 80 years ago, and has since become commercially known for biological pest control. B. thutinglensis spore-forming cells each produce a spore (endospore) and a diamond-shaped protein crystal (paraspore or inclusion bodies). Emtomocidal properties are attributed

exclusively b-endotoxin, which is the main component of the paraspore crystal. When the crystal becomes soluble in the gut of the insect, it causes the formation of a proxin, which is activated as a result of proteolytic cleavage.

Being a rather specific species with respect to Lepidoptera insects and some flies and bridges, B. thuringiensls is harmless to the insect resistant insects, animals and humans. Currently in the USA. B. thuringiensls of the kurstahl variety includes the B. thurlngiensis insecticidal products most commonly used to control these pests. One W00

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however, these products are not effective enough in industrial applications.

Synthetic pyrethroids, on the other hand, are the chemical insecticides most widely used to control lepidopteran pests, pyrethroids are highly effective and provide a stronger pest control than the currently used B. thuringiensls insecticides, However, the disadvantage associated with the use of pyrethoids is that they are toxic to unreachable invertebrates, thereby presenting a hazard to the surrounding water environment. food. Therefore, there is a need for effective Lepidopteran insecticides that are environmentally friendly, cost effective and that duplicate the effectiveness of synthetic pyrethroids.

An object of the invention is to provide an insecticidal preparation based on a new strain of B. thurirtglensis Var, morrisoni NRRL B-18196 for killing insect species of the order Lepidoptera, in particular Trichqpiuslanl, cabbage moth, and Artogela garay; / Imported goods

A new B. thuringiensls strain was deposited in a crop collection at the North American Department of Agriculture Regional Research Center, Peori, Illinois 61604, under registration number NRRL B-18196. The progeny of this strain will be referred to as NRRL.

The new strain is a sprogenic, crystal-forming mutant strain isolated from environment B, thuring iensis and has the ability to produce bipyramdal crystals consisting of a toxic protein and is unique in that it is auxotrophic for leucine and valine for growth, sporulum phase and crystal production. In contrast, previously observed strains of B. thuringiensis showed auxotrophy, which was satisfied without the amino acid, K.W. Nickercon et al, Appl, Microblot, 28, 124-128 (1974).

The term auxotrophy is defined here as meaning the nutrient requirement for growth, sporulation, and production of microorganism crystals.

The phrase amino acid complex of a culture medium is defined herein to mean a culture medium containing a combination of two or more amino acids.

The new strain NRRLB-18196 was propagated from the soil layer using a non-classical method of selection using sodium acetate and tepe. In accordance with this method, the aqueous nutrient medium is buffered to about 0.25 M sodium acetate to allow only undesired spore-forming organisms to emerge,

present in the environmental sample

soil layer. Then grown

spore-forming organisms and non-spore-forming organisms in the sample were killed by heat treatment. The surviving spores were then plated on plates and grown on an appropriate agar culture medium to produce new strains of the invention. Without resorting to any particular theory, it is contemplated that new strains of the invention are formed during the selection process as a result of mutagenesis naturally occurring in strains

B.thuringiensfs, followed by treatment with sodium acetate and heat.

An example of a selection method is as follows: 0; 5 g of soil was added to 10 ml of L-broth (100 ml of water, 1 g of tryptone,

0.5 g of yeast extract, 0.5 sodium chloride) in a 125 ml baffle. L-broth was buffered to 0.25 M sodium acetate. The mixture was shaken for 4 hours at 250 rpm and 30 ° C. After using a flowing thermal pasteurization device, the mixture was heated at 80 ° C for 3 minutes. Heat treatment mixture was plated on plates with L-agar (L - broth, (approved with 1.5% agar)

and incubated overnight (1-6 hours) at 30 ° C.

Strain NRRL B-18196 has the following characteristics: colonies are round, solid, convex and cream in color with a colony diameter of 2-7 mm on nutrient agar after 24 hours. Vegetative cells are aerobic, gram-positive, motile bacilli

1-1.2 microns in size by 3-5 microns. The terminal (apical) spores of vegetative cells do not inflate sporangia and make up from 33 to 50% of vegetative bacilli. Cells are positive for catalase, glucose enzyme, casein hydrolysis, but contrary to the taxonomic description of previously observed B. thuringiensis strains, the strains of the invention do not use citrates. The new strain also does not ferment mannitol, erabinose and xylose.

Additionally, NRRL B-18196 strain hydrolyzed starch, fermented sucrose and produced lecithinase; the cells didn’t use esculin without producing- / and urease, did not ferment salicin and V. 1; Tamm is resistant to Ј penicillin-type antibiotics, such as naficillin, ampicillin and methicillin, and is resistant to (low levels, i.e. near South / ml, neomyne, otherwise, kanamycin and ethide bromide.

As mentioned above, the production of pores and protein crystals by B. thuringiensls strain having the characteristics of NRRL B-18196 requires an aqueous nutrient redia that contains a leucine-valine-like amino acid complex. For optimal crystal formation by the bacterium according to the invention, the preferred composition of a special nutrient medium is as follows: 0.3% tryptone, 0.2% riptose, 0.45% yeast extract, 0.01M added in sodium phosphate buffer at pH 6.8 with the addition of 10 MgS04 MnS04 after autoclaving. The strain is grown at a temperature of 0 ° C at 250 rpm in 2 liter Erlenmeyer flasks with triple septa with a volume of 25% and stored at a temperature of 25 ° C in a medium of TK, cured 1.5 % agar.

Another suitable nutritional: redid having the desired amino acid complex can be used to proliferate bacteria according to the invention. However, a different nutrient medium used than the preferred nutrient medium can lead to reduced crystal formation.

Cell production is carried out under aerobic conditions at any temperature satisfactory for the growth of microorganisms according to the invention, i.e. from about 10 ° C to 40 ° C; preferred temperature range is between 27 ° C and 32 ° C. The pH of the culture medium suitable for growing a B. thuringlensls culture is close to neutral, i.e. pH 6.7 to 7.2. The incubation time is the time required for complete release of the spores and the crystals are preferably 18 to 24 hours. Cells can be grown in any shake deflector flask in a series of small experiments. For larger scale operations, it is convenient to grow the culture in the reservoir using stirring and aeration in relation to the inoculated liquid culture medium. After incubation, cells are harvested by the traditional sedimentation method, such as by centrifugation or filtration. Cells can be used as such or in frozen form for subsequent use.

To obtain an insecticidal composition comprising a strain of B. thurlngiensis var. morrisoni NRRL B-18196 as an active ingredient, a strain that constitutes

0 to 30% of the total volume is mixed with inert ingredients which make up the residual volume, i.e. at least 70% of the total. An inert ingredient may simply be a diluent, for example water.

Other inert ingredients may include preservatives, flow control agents, adhesives, emulsifiers, sawing agents,

0 surfactants, fillers and buffer. The dosage range used is 1.1 x 1010 to 5.1012 spores per 0.405 ha. An insecticide containing the mutant B. thuringlensls var morrisoni NRRL B-18196 a

5 as an active ingredient in the indicated doses, it is highly effective in killing insects of the order Lepidoptera, in particular cabbage moths and imported meringitides (see table below).

0 An example of a method for producing an insecticidal preparation involves mixing together using an air mill to ensure particle size and homogeneity of strain NRRL B-18196 of

5 to 30% of the total volume with inert ingredients that make up the remaining volume. These inert ingredients include a preservative, two surfactants, a flow control agent, a diluent, and a buffer. The mixture is then granulated using a compression technique and packaged in airtight, moisture-free containers. Insecticides can be sprayed with 5 methods using traditional techniques and spray devices. The new insecticide may also be encapsulated or encapsulated in an appropriate encapsulating material, such as an organic polymer, and used in powder form. It is also contemplated that the genetic material for the b-endotoxin of the new B. thurlngiensis strain could be. transferred using recombinant DNA technology to other bacteria and plants to provide enhanced pest control.

The following example is intended to illustrate the invention and does not limit the scope of claims as defined by the claims.

In field trials, the efficacy of an insecticide containing strain NRRL B-18196 as an active ingredient against cabbage moths and imported mermitides was compared with the efficacy of a synthetic insecticidal product containing pyrethroid, PIDRIN and B. thuringiensis insecticidal product. Varieties of Kurstaki MDIPEL is PIDRI is a product mark sold by Shell Corporation and contains 30% cyano (3-phenoxyphenyl) methyl-4-chloro (1-methyl) tyl) benzene acetate and 70% inert ingredients. DIPEL is a trademark of a product sold by Abbott Laboratories and contains 3.2% B. thuringiensis species of Kurstaki and 98.8% of inert ingredients.

Two other new insecticidal preparations were also tested containing B. thuringiensis varo Russtarl NRRL B-18197 and B-18195 strains as active ingredients. Insecticides based on the new NRRL strains B-18197, B-18195 and B-18196 contained 5.5 x 10 spores per 0.5 L of water, 0.01% of the total volume was liquid detergent (added as a spraying agent) and the rest the volume was water.

On the east coast of Maryland, leaf cabbage was directly planted with seeds in 4 rows of 609.60 cm daina on July 07, 1986. Plants were 5.08 cm apart and 91.44 cm apart from each other. Processing was organized according to the scheme of randomized blocks with 4 repetitions. Each row was buffered by a guard row. The soil was sandy loam soil Norfolk A. All spray treatments were 5.5 x 109 spores per 0.5 liters of water, to which 0.01% liquid detergent was added as a sawing agent. Sprayings were carried out using a garden trombone type sprayer calibrated to deliver 113.55 L of 0.405 ha.

The series were sprayed on August 11, 18 and August 25 and 2 September and 8 September. On September 15, damaged foliage and the number of insects were estimated. Insect pressure due to a naturally occurring invasion of cabbage scoops and imported meringitides Oylo moderate. Foliage damage scores were indexed as follows:

1) 0-3% damage - random holes on the leaves;

2) 4-10% of damage - several leaves with holes;

3) 11-25% of damage is the average / moderate number of leaves with holes;

4) 26-50% of damage - most leaves with holes; and

5) 51-100% damage - damage to the root neck and / or all leaves with holes.

The data were analyzed by analysis of variance, and the average values

were determined by testing the Duncan Multiple Series (DHRT) at P level 0.05 (Duncan 1951). The results are summarized in the table.

The table clearly shows 10x

an increase in the insecticidal activity of an insecticide containing NRRL B-18196 as an active ingredient in comparison with the commercial product Dipel.

This increase was well demonstrated when, after treatment with Dipel, the level of damage was

less than 10%, which corresponded to the processing of NRRL B-18196 at a dilution of 1:10.

It is understood that modifications and changes may be made to the above description without departing from the spirit and scope of the invention.

Claims (3)

SUMMARY OF THE INVENTION Bacillus thuringiensis var. Kurstaki NRRL B-18196 for the preparation of an insecticidal preparation. 1, Any two numbers in the same column followed by the same letter differ significantly (P 0.05) DMRT 2. Spraying was carried out on August 11,18,25 and 2,8 September br. | 3. The harm caused mainly by the scoop of cabbage and imported mermitides, I 4 DIPEL 7.26 billion international units for 373.2 hours | 5 Calibrated by the output of 7.26 billion international units n 373.2 h,